The techniques of plant biotechnology have improved during the last ten years so that most of the crop species, which are of importance to mankind, can be routinely transformed. The industry seeks for new traits not only for agricultural or nutritional purposes, but also for pharmaceutical purposes. There is an increasing interest to develop efficient and economic production systems for useful biological compounds. Transgenic plants play an important role in the research aiming to develop such a system. Given the concerns of environmental impacts of genetically modified crops this development has clearly created a need for a reliable system to prevent transgene flow among crops and in their relatives. Accordingly, several research groups around the world are currently engaged in developing techniques for gene containment in transgenic crops.
The technologies that are aimed to prevent transgene flow can be categorized into one-component and two-component technologies. The main feature of the one-component systems is a factor, which enables a negative selection of transgenes from plant populations. As examples, the known concepts of male sterility, chloroplast transformation or ‘Terminator’ technology, are mentioned. One-component systems decrease gene flow, but they do not always provide a completely reliable containment. Two-factor technologies were developed in order to improve the gene containment. These systems use negative selection factors together with a recovering (rescuing or repairing) factor. The negative selection factors are usually lethal for the plant and therefore they totally prevent the transgene flow. The rescuing factor represses the action of the negative selection factor, disrupts its function or recovers the functions blocked by the negative selection factor. Examples of two-factor technologies are the systems described in the International patent publications WO 94/03619 (Bright et al.) and WO 00/37660 (Fabijanski et al.).
The International patent publication WO 02/064801 (Kuvshinov et al.) describes a two-factor system, where an excision construct (EC) is linked to the transgene of interest (TGI). The EC excises the whole insert from the genome of the host organism under natural conditions. An artificially activated repression construct represses the action of the EC and saves the transgenic insert in the host genome. This system removes the entire transgene insert and leaves the host genome free from the foreign genes. Thus, in natural conditions transgenic plant produces non-transgenic seeds only and can not produce transgenic seeds.
According to Gressel, Trends Biotechnol., 17, 361-366, 1999, inactivation of negative selection factor (gene) may happen with a frequency of approximately 10−6. In practice this means once during a growth season, in each middle sized field plot. Such a frequency of gene escape from a field, where the transgenic crop is cultivated for production of a vaccine or other pharmaceutical compounds would create public concerns. It has been suggested that the inactivation problem may be solved by using an one-component concept called mitigation tandem technique. In this technique the desired transgene is coupled in tandem with gene(s) that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Examples of features that could be used in mitigation technique are secondary dormancy and dwarfing. A problem encountered with the tandem mitigation technique is that due to absence of a recovering system, removal of transgene from the population demands several generations. Therefore this technique does not provide sufficiently reliable transgene containment. The scarce sources of genes capable of mitigating, is another limitation of the technology.
The International patent publication WO 02/06498 corresponding to the US patent applications U.S. Ser. No. 10/892,513, U.S. Ser. No. 10/644,664 and U.S. Ser. No. 09/617,543 (Kuvshinov et al.) all disclose a two-factor system called RBF-system (recoverable block of function system), which comprises at least one blocking construct (BC), which is an insert consisting of a blocking gene, which is linked to a transgene of interest (TGI), which is a gene encoding a desired protein or gene product, and at least one recovering construct (RC). According to said disclosure the BC(s) block(s) a vital physiological or molecular function of the host plant through developmental or organ specific expression. The RC is induced by an externally controllable stimulus and when induced it recovers the function previously blocked by expression of the BC(s).
Due to the increasing use of transgenic plants, not only new methods for controlling transgene escape in plants are needed, but also alternative new blocking genes, which may block essential functions of the plant particularly during germination and which can be recovered by a user controlled intervention.
Phytoene synthase is an enzyme in the biosynthetic pathway (FIG. 1) leading to production of carotenoids, which are biologically important in many organisms ranging from bacteria and fungi to higher plants.
Phytoene synthase produces phytoene (C40) from geranylgeranyl diphosphate (C20), which is a mutual precursor of carotenoids, tocopherols, gibberellins and chlorophyll (Fray et al., Plant J., 8, 693-701, 1995; Shewmaker et al., Plant J., 20, 401-412, 1999; Sandmann, Trends in Plant Sci., 6, 14-17, 2001). Bacterial crtB genes encoding phytoene synthase have been expressed in plants in order to increase content of carotenoids. “Golden” Rice (Oryza sativa) is an example of a transgenic plant seed overexpressing the phytoene synthase (Beyer et al., J. Nutr., 132, 505S-510S, 2002).
Seed-specific expression of the gene encoding phytoene synthase leads to a 50-fold increase in carotenoids, decrease in chlorophyll levels and slight delay of seed germination in Brassica napus (Shewmaker et al., Plant J., 20, 401-412, 1999). In tomato (Lycopersicon esculentum), constitutive expression of the crtB gene under 35S promoter results in a decreased level of chlorophyll and dwarfism, which was provoked by 30-fold reduction in levels of gibberellin (GA) (Fray et al., Plant J., 8, 693-701, 1995).
Embryo specific overexpression of plant endogenous phytoene synthase results in increased levels of carotenoids in seeds of Arabidopsis (Lindgren et al., Plant Physiol., 132, 779-785, 2003). The plant derived phytoene synthase increases the level of chlorophyll, whereas the level of α-carotene is only slightly increased. The plant derived phytoene synthase also results in decreased levels of gibberellins, whereas an increased level of abscisic acid (ABA) leads to delayed germination, which is not recoverable by a gibberellin addition.
Even if it is known that constitutive, seed or embryo specific overexpression of phytoene synthase delays seed germination, the gene encoding phytoene synthase does not totally prevent germination, because expression does not occur during germination. Because germination is delayed and not fully blocked, the seedlings are capable of overcoming the lack of gibberellins and excess of ABA. Accordingly, the constitutive, seed and embryo specific expression of crtB gene is not suitable for developing RBF-systems for controlling of transgene escape.